tRNA Arg binds in vitro TDP-43 RNA recognition motifs and ligand of Ate1 protein LIAT1

Transactive response DNA-binding protein 43 (TDP-43) is important for RNA metabolism in all animals and its malfunctions are linked to neurodegenerative and myodegenerative diseases in humans. Arginyl transferase Ate1 transfers an arginyl group from arginylated tRNA Arg to proteolytic fragments of the C-terminal region of TDP-43, prompting their degradation by the ubiquitin proteasome system, thus contributing to TDP-43 proteostasis. To gain more insight into the molecular basis of TDP-43 arginylation, we tested if tRNA Arg could bind in vitro to a panel of recombinant multidomain constructs of human TDP-43 or to the arginylation cofactor protein LIAT1. We observed that in vitro- transcribed human tRNA Arg directly interacts with the RNA recognition motifs of TDP-43 and that their binding is stabilized by dimerization, which is promoted by the amino-terminal domain and the nuclear localization signal sequence of TDP-43. Moreover, the same human TDP-43 constructs that bind tRNA Arg bind native fungal tRNA Phe , suggesting that TDP-43 can bind different populations of tRNAs. Interestingly, human tRNA Arg is also able to bind recombinant mouse LIAT1 suggesting, for the first time, that LIAT1 is an RNA-binding protein. Our findings open a new perspective on the intricate crosstalk between protein and tRNA metabolism, which may eventually contribute to the understanding of the role of TDP-43 proteostasis in health and disease.


Description
Post-translational Nt-arginylation is the non-ribosomal transfer of an arginyl group from arginylated tRNA Arg to target proteins that bear an amino terminal aspartate, glutamate or oxidized cysteine.This modification occurs in all eukaryotes and is catalyzed by the arginyl-tRNA Arg protein transferase Ate1 (Manahan and App, 1973;Balzi et al., 1990;Hu et al., 2006).Ntarginylation may direct proteins to degradation by the ubiquitin proteosome system (Ciechanover and Kwon, 2015;Varshavsky, 2019) and by autophagy (Jiang et al., 2016), or it may influence protein dimerization and intracellular localization (Sambrooks et al., 2012).Nt-Arginylation has neuroprotective and neuroregenerative effects (Bongiovanni et al., 1995;Ingoglia, 2015;Ingoglia and Jalloh, 2016) and has been shown to influence the folding and proteostasis of neuronal proteins important in human neurodegeneration such as amyloid beta, synuclein and TDP-43.In particular, TDP-43 is important for RNA splicing and regulation of translation (Freibaum et al., 2010;Buratti and Baralle, 2012;Bhardway et al., 2013).In humans, TDP-43 is involved in fatal neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration FTLD (Neumann et al., 2006), as well as in myodegenerative diseases such as sporadic inclusion bodies myositis sIBM (Cortese et al., 2014) and myoclonic epilepsy associated with ragged red fibers MERRF (Mancuso et al., 2004;Mori et al., 2019).Upon prolonged cellular stress, TDP-43 translocates from the nucleus and becomes increasingly cytoplasmic, proteolytically fragmentated, hyperphosphorylated and polyubiquitinated, and aggregates in insoluble cytoplasmic inclusions.Progranulin mediates caspase-dependent fragmentation of TDP-43 generating a heterogenous population of C-terminal fragments (CTFs) of ~ 25 kDa and ~35 kDa that have been found in the cytoplasmic inclusions of some FTLD and ALS patients as well as in model animals (Neuman et al., 2006;Zhang et al., 2007;Igaz et al., 2008;Li et al., 2015).A decrease in Nt-arginylation of the CTFs of human TDP-43 has been shown to increase their aggregation in the cytoplasm (Brower et al. 2013;Kasu et al. 2018) which may contribute to the imbalance of intracellular TDP-43 and related proteinopathy.
At a molecular level, TDP-43 is a dimeric protein prone to oligomerization (Mompean et al., 2017;Afroz et al., 2017;Wang et al., 2018) and it consists of a conserved region composed of an amino-terminal dimerization domain (NTD), a nuclear localization signal (NLS) and two tandem RNA recognition motifs RRM1 and RRM2 followed by a non-conserved glycinerich carboxyl terminus of low structural complexity (Laurents et al., 2021;Capitini et al., 2021;Figure 1A).To further the thorough understanding of the molecular basis of TDP-43 Nt-arginylation, we used an in vitro system to transcribe human tRNA Arg containing also a 3' CCA trinucleotide (htRNA Arg ) and tested its interaction with a set of recombinant constructs of human TDP-43 (hTDP-43) consisting of one or more of the features in the conserved region.The constructs produced were hTDP-43 NTD (residues 1-85), NLS-RRM1-RRM2 (residues 85-266), RRM1-RRM2 (residues 101-266), and a truncated construct NTD-ΔNLS-RRM1-RRM2 (residues 1-85 and 101-266) lacking the NLS linker because it is a hotspot for proteolytic attacks by host proteases.NTD, NTD-ΔNLS-RRM1-RRM2 and NLS-RRM1-RRM2 were purified as dimeric proteins, whilst RRM1-RRM2 was monomeric.When tested in vitro for binding to htRNA Arg , we detected stable complex formation with NLS-RRM1-RRM2 (Figure 1B Left), RRM1-RRM2 (Figure 1C Left) and NTD-ΔNLS-RRM1-RRM2 (Figure 1D Left).We did not observe that hTDP-43 NTD on its own could form a stable complex with htRNA Arg .The hTDP-43 NLS-RRM1-RRM2:htRNA Arg complex was very stable, unaffected by doubling the physiological concentration of KCl or by high concentrations (up to 20%) of PEG 8000 used as a macromolecular crowding agent.The RRM1-RRM2 complex with htRNA Arg contained less htRNA Arg than the one with NLS-RRM1-RRM2 (Figure 1E).Besides binding to htRNA Arg we also observed successful binding of these constructs to native yeast tRNA Phe (ytRNA Phe ; Figure 1B, 1C, 1D Right), suggesting that general tRNA binding could be a property of the hTDP-43 central region.Increasing the concentration of hTDP-43 NLS-RRM1-RRM2 or NTD-ΔNLS-RRM1-RRM2 resulted in higher band shifts of both htRNA Arg and ytRNA Phe ; such migration pattern could be explained by protein or RNA concentration-dependent oligomerization and/or formation of different types of protein:tRNA complexes.Mammalian Ate1 co-factor protein LIAT1 can enhance in vitro Nt-arginylation (Brower et al., 2014).Vertebrate LIAT1 proteins consist of conserved amino terminal and central regions which contain a glutamate-rich region, a lysine-rich region, and a ~30 residue-long Ate1-binding region termed the LIAT1 domain, and a variable carboxyl-terminal region (Brower et al., 2014).Sequence analysis suggested that both human and mouse LIAT1 are intrinsically disordered proteins (Brower et al., 2014;Arva et al., 2020) as supported by their AlphaFold models, AF-Q6ZQX7 and AF-Q810M6 (Jumper et al., 2021).We checked if LIAT1 could bind htRNA Arg .For interaction assays, we opted to use mouse LIAT1 (Figure 1F) because it is the shortest mammalian LIAT1 that was shown to interact directly with Ate1 (Brower et al., 2014;Arva et al., 2021).Recombinant mouse LIAT1 (mLIAT1) produced in Escherichia coli, purified in aerobic conditions as a soluble light brown protein.The construct eluted in size exclusion chromatography as a ~48 kDa protein in agreement with the theoretical molecular weight of dimeric mouse LIAT1 (Figure 1G).mLIAT1 migrated on SDS-PAGE gels as a ~30 kDa protein (Figure 1G) which is bigger than expected for a protein of ~24 kDa theoretical molecular weight.This anomalous migration might be explained by the presence of a ligand, intramolecular disulfide bridges, detergent binding or by being intrinsically disordered.Sequence analysis of mouse LIAT1 by HeMoQuest (Paul George et al., 2020) predicts the existence of a transient hemebinding site (residues Cys122 and Cys197) and the presence of disulfide bonds.The latter is supported by the presence of a CXXC motif (Cys194 and Cys197), which in some redox proteins regulates disulfide bond formation and dimerization (Chievers et al., 1996).These predictions could explain the brownish color of mLIAT1, as well as its oligomerization and anomalous migration in SDS-PAGE.We note that mouse LIAT1 also contains a putative bipartite nuclear localization signal sequence (amino acids K 49 RKVKKKKKKKKTKG 63 ) that is conserved in different vertebrate LIAT1 proteins (Figure 1H).The presence of this signal could explain a previous observation of mouse LIAT1 being found in both the nucleus and the cytosol (Arva et al., 2021).We observed in vitro interaction between mLIAT1 and htRNA Arg resulting in four different protein concentration-dependent mLIAT1:htRNA Arg complexes (Figure 1I).This result suggests the coexistence of different mLIAT1 oligomers which bind separately to htRNA Arg , and this is in accordance with a previous study that reported LIAT1 selfoligomerization in vivo (Bower et al., 2014).
We could not analyze if the full-length human TDP-43 or its CTFs, human LIAT1 or human Ate1 interact with htRNA Arg , because of problems encountered in the recombinant production of these constructs.These difficulties may be due to unmet folding requirements.In this regard, we note that in both the three-dimensional structures of mouse TDP-43 RRM2, PDB 3D2W (Kuo et al., 2009) and of yeast ATE1, PDB 7WG4 (Kim et al., 2022), there is a cis prolyl peptide bond (Figure 1J).In the future, it would be of interest to identify if and which trans-cis peptidylprolyl isomerases are required for successful human TDP-43 and Ate1 folding.
In summary, human TDP-43 multidomain fragments that include the RRMs were observed to bind efficiently to in vitrotranscribed human tRNA Arg .In vivo, TDP-43 CTFs are heterogenous in length, and often contain parts of the RRMs -e.g.TDP-43 CTF 219-414 and 247-414 (Bower et al., 2013;Li et al., 2015;Kasu et al., 2018).These RRM-containing CTFs might interact with tRNA Arg , influencing binding specificity and Nt-arginylation kinetics and contributing to the self-regulation of TDP-43 proteostasis and pathology.We also observed that the RRM-containing constructs of TDP-43 that bind tRNA Arg are also able to bind to native fungal tRNA Phe .The interaction between TDP-43 and tRNA Phe could be of physiological relevance given that a point mutation of human tRNA Phe causes MERRF myopathy characterized by cytoplasmic inclusions containing TDP-43 (Mancuso et al., 2004) and that aminoacylation of tRNA Phe is compromised in ALS patients (Malnar Črnigoj et al., 2023).Moreover, a previous report showed that in vivo recombinant polyhistidine-tagged human TDP-43 can also bind mitochondrial tRNA Asn , tRNA Gln and tRNA Pro (Izumikawa et al., 2017).Taken together, this report and our data suggest that TDP-43 may be involved in the metabolism of tRNAs in general.Finally, we observed strong binding of recombinant mouse LIAT1 to htRNA Arg , a capacity that could extend to other LIAT1 sequelogues.In addition to binding to Ate1, mouse LIAT1 interacts with bifunctional arginine demethylase and lysine hydroxylase jumonji domain-containing protein 6 (JMJD6).JMJD6 is involved in RNA splicing and, together with ribosomal proteins RPS14 and RPS19, in tRNA binding and ribosome biogenesis (Bower et al., 2014;Arva et al, 2021).Noteworthy, a jumonji domain-containing hydroxylase protein TYW5 binds and modifies human tRNA Phe (Noma et al., 2010;Kato et al., 2011).Given that the partner proteins of LIAT1 are involved in different aspects of RNA metabolism, it is foreseeable that LIAT1 capacity to bind RNA could have functional implications which may reflect also on TDP-43 proteostasis.
Yeast phenylalanine tRNA (ytRNA Phe ) was purified from yeast (Merck) and resuspended to 1 mg mL -1 in SEC buffer, renatured (treated 65°C for 90 seconds and then allowed to slowly cool down to room temperature inside lab heating block) and applied on a HiLoad Superdex 75 16/60 column in SEC buffer; eluted peak fractions were quantified assuming an extinction coefficient ε = 560 000 M -1 cm -1 at 260 nm.
Electromobility shift assays (EMSA) were performed on 6% polyacrylamide gels (1 mm x 100 mm x 80 mm), in 0.5 x TBE, at 80V, 20°C, for 70 min.30 μL reactions were set mixing increasing amounts of recombinant protein (0.5 -32 μM final concentration in reaction, assuming total protein concentration as of monomeric protein) with fixed amount of tRNA (0.5 μM final concentration in reaction) in SEC reaction buffer supplemented with 5% PEG 8000, adding tRNA as the last component and incubation time was 30 min at 20°C, after what native gel loading buffer (2.5xTBE, 50 % glycerol, 0.1 % blue bromophenol, 0.1 % xylene cyanol) was added and 15 μL of sample were loaded.Gels were stained for 15 min with nucleic acids binding chelating agent SYBR safe (EuroClone) diluted in 0.5xTBE, washed in 0.5xTBE and visualized on UV 260 transilluminator.ImageJ (NIH) was used to quantify unbound fraction of loaded tRNA and plotted using Excel; the fraction of bound tRNA was obtained by dividing difference of total loaded tRNA and unbound tRNA with total tRNA.PyMol (DeLano, 2020) was used for 3D models visualization and analysis and for generating structural figure; Clustal Ω (Sievers et al., 2012) was used for multiple sequence alignment; Motif Scan (Gasteiger et al., 2003) was used for protein sequence analysis.